Membrane thickness affects ELIC activity & M4 helix structure

Pentameric ligand gated ion channels (pLGICs) are a superfamily of ion channels involved in fast neural transmission. Some examples include the GABA(A) receptor and nicotinic acetylcholine receptor (nAchR). In the nAchR, it has been shown that thick membranes promote coupling of agonist binding to channel activation. However, the effect of membrane thickness on the ion transport activity of any pLGIC is not known. To investigate this, we reconstituted the model pLGIC, Erwinia ligand gated ion channel (ELIC), in liposomes consisting of di-monounsaturated phosphatidylcholine (PC) of varying acyl chain length (16-22), and assayed ion flux using a sequential mixing fluorescence quenching assay. ELIC exhibited maximal agonist responses in di-20:1PC liposomes with a dramatic decrease in thinner or thicker membranes. To verify that differences in agonist responses are not a function of changes in channel orientation in the liposomes, we further assayed an ELIC construct with a quenchable fluorescent label in the N-terminal ECD. Minimal differences in the amount of outwardly facing channels were observed in different membrane conditions confirming that changes in agonist responses are primarily a function of channel activity. To investigate the structural basis of ELIC sensitivity to membrane thickness, we obtained apo and agonist-bound structures of ELIC in a circularized nanodisc with di16:1PC, di20:1PC or di22:1PC using single particle cryo-EM. In the agonist-bound structures, the membrane facing helix, M4, is poorly resolved in thin (di16:1PC) and thick (di22:1PC) membranes but clearly resolved in di20:1PC, indicating a correlation between the structure and dynamics of M4 and the optimal membrane thickness that supports channel function. These findings indicate that ELIC function is promoted by thicker membranes and suggests that sensing of membrane thickness is mediated by M4, as has been proposed for the nAchR.